Method and composition for treating paramyxovirus

ABSTRACT

The present invention relates to a method of treating respiratory syncytial virus (RSV) through the exogenous administration of recombinant or natural interferon-beta (IFN-β) to lung epithelial cells. Preferably, IFN-β is administered to hosts suffering from RSV in effective nebulized or aerosolized doses.

FIELD OF THE INVENTION

The present invention is directed to a method of treating respiratorydiseases caused by respiratory syncytial virus (RSV) through theexogenous administration of recombinant or natural interferon-beta(IFN-β). More particularly, the invention relates to methods fortreating RSV through the administration of an effective amount of IFN-βto resident lung cells (e.g. macrophages, epithelial, etc.) to reduceRSV replication and/or to prevent infection of adjacent cells.Preferably the IFN-β is administered through nebulization. The nebulizedcompound is inhaled by the patient so as to activate lung cells tointerfere with virus replication. It has been found that IFN-β interactswith appropriate receptors on epithelial cells in mammals to restrictRSV replication. The invention also provides pharmaceutical compositionssuitable for inhalation and for the purposes indicated above.

BACKGROUND OF THE INVENTION

Paramyxoviruses comprise a family of RNA viruses tropic for the humanrespiratory tract, that result in approximately 13 million infectionsper year. RSV, a major pathogen within the Paramyxovirus family, causessevere lung disease in young children, elderly adults, andimmunocompromised individuals. To date, no effective vaccine for RSVpresently exists despite the estimated 1 million deaths this viruscauses annually in infants and children.

It has been found that most infected older children and adults restrictand eliminate RSV rapidly. However, young infants and children aresusceptible to infection of bronchiolar and alveolar cells resulting inbronchiolitis, pneumonia or respiratory compromise which may result indeath. Symptoms such as nasal discharge, fever, fatigue, deep cough,wheezing and shortness of breath are generally associated with RSVinfections. Reinfections are common, especially in infants and youngchildren.

Structurally, RSV is an enveloped, negative stranded RNA virus of thefamily Paramyxoviridae and of the genus pneumovirus. The two majorenvelope proteins are the G protein, which is believed to be responsiblefor attachment of the virus to the host cell membrane, and the fusion(F) protein, which is believed to be responsible for fusing the virusand cell membranes. Virus-cell fusion is a necessary step for infection.F protein is required for cell-cell fusion which is another way tospread the virus from an infected cell to an adjacent uninfected cell.Antibodies directed against these proteins do not confer significantimmunity in humans.

The targets for RSV infections are generally the terminal bronchiolesand alveoli in mammals which are lined by lung epithelial cells andalveolar macrophages. If these cells permit unrestricted RSVreplication, viral burden progressively increases resulting indenudation of the airway and destruction of alveolar macrophages whichdefend the lung against other pathogens.

It has been found that most children can recover from RSV infectionindicating that they can eliminate RSV. This implies that intrinsiccellular mechanisms, as yet undefined, may restrict virus replication.However, the reason why some children have progressive disease that canculminate in overwhelming infection and death is not yet known.

Although RSV has been extensively studied, there are no availableeffective vaccines to combat RSV. The lack of an effective vaccine forRSV suggests that strategies to augment intrinsic lung defenses againstthis virus could offer potential clinical benefits. Therefore,applicants examined whether differentiated human lung epithelial cellspossess intrinsic mechanisms to restrict RSV replication, whether suchintrinsic mechanisms could be augmented by anti-viral cytokines (e.g.IFN-β), and whether anti-viral cytokines could be operative beforeinduction of humoral or cell-mediated immune responses.

In this regard, applicants studied RSV replication in normal human lungepithelial cells transformed with an origin defective SV-40 vector, i.e.9HTE (tracheal origin) and BEAS 2B cells (bronchiolar origin). Inaddition, applicants examined the non-transformed human A549 cells(alveolar epithelial origin) derived from an alveolar carcinoma. Thesedifferentiated lung epithelial cell lines offer an in vitro model withwhich insights into the molecular mechanisms that restrict RSV in humanlung cells can be examined. In addition, the differentiated lungepithelial cell lines offer advantages over highly permissive celllines, such as CV-1, HEp-2 cells, or HeLa cells, which lackcharacteristics of differentiated human lung cells.

Moreover, it is believed that alveolar macrophages may have an importantrole in restricting replication of respiratory viruses through theircapacity to produce tumor necrosis factor a (hereafter referred to asTNFα). Local production of TNFα at the site of virus infection haspreviously been shown to restrict vaccinia virus replication (Sambhi,S.K., Kohonen-Corish, M. R. J., and L. A. Ramshaw. 1991. Localproduction of tumor necrosis factor encoded by recombinant vacciniavirus is effective in controlling viral replication in vivo. Proc NatlAcad Sci. 88:40254029). In vitro and in vivo studies have demonstratedthat alveolar macrophages are permissive to RSV infection/replicationand produce TNFα following RSV infection (Panuska, J. R., Hertz, M. l.,Taraf, H., Villani, A., and N. M. Cirino. 1992. Respiratory syncytialvirus infection of alveolar macrophages in adult transplant patients.Am. Rev. Respir. Dis. 145:934939).

Furthermore, it has been determined that TNFα restricts further RSVreplication in alveolar macrophages through an autocrine mechanism. Theintimate contact between alveolar macrophages and epithelial cells fromterminal bronchioles and alveoli suggests that TNFα could potentiallyrestrict RSV through paracrine mechanisms. However, the potential use ofrecombinant TNFα as a systemic antival therapy is limited by itstoxicity.

Applicants therefore examined whether RSV could be transmitted betweenlung epithelial cells and alveolar macrophages. Applicants furtherexamined TNFα binding to lung epithelial and CV-1 cells and whetherthese separate cell types expressed the 55 and 75 kDa TNFα receptorsubtypes (Loetscher, H., Pan, Y. E., Lahm, H., Gentz, R., Brockhaus, M.,Tabuchi, H., and W. Lesslauer. 1990. Molecular cloning and expression ofthe human 55 kd tumor necrosis factor receptor. Cell 61:351-359;Pennica, D., Lam, V. T., Mize, N. K., Weber, R. F., Lewis, M., Fendly,B. M., Lipari, M. T., and D. V. Goeddel. 1992. Biochemical properties ofthe 75-kDa tumor necrosis factor receptor. J. Biol Chem. 267:21172-21178). The 55 kDa TNFα receptor mediates the anti-viral effects of TNFα insome (Wong, G. H. W., Tartaglia, L. A., Lee, M. S., and D. V. Goeddel.1992. Antiviral activity of tumor necrosis factor (TNFα) is signaledthrough the 55-kDa receptor, type 1 TNF.J. Immunology 149:3350-3353),but not all cell types (Rothe, J., Lesslauer, W., Lotsher, H., Land, Y.,Koebel, P., Kontgen, F., Althage, A., Zinkernagel, R., Steinmetz, M.,and H. Bluethmann. 1993; Mice lacking the tumor necrosis factor receptor1 are resistant to TNF mediated toxicity but highly susceptible toinfection by Listeria monocytogenes. Nature 364:798-802).

In this regard, it is known to some degree that TNFα and IFN-β,individually, or synergistically, can restrict replication of both RNAand DNA viruses (Sen, G. C., and R. M. Ransohoff. 1993.Interferon-induced antiviral actions and their regulation. Adv. VirusRes. 42:57-101; Wong, G. H. W., Kamb, A., and D. V. Goeddel 1993.Antiviral properties of TNF. In B. Beutler, editor. Tumor NecrosisFactors: The Molecules and Their Emerging Role in Medicine.Raven Press.Ltd, New York, 371-381). IFN restricts RSV replication in lungfibroblast cell lines (Moehring, J. M., and B. R. Forsyth. 1971. Therole of the interferon system in respiratory syncytial virus infections.Proc Soc Exp. Biol Med.138:1009-1014) but its effects on RSV replicationin lung epithelial cells had not been previously examined. RSV induceslow levels of IFN in only ˜50% of infected children (Hall, C. B.,Douglas, R. G. Jr, Simons, R. L., and J. M. Geiman.1978. Interferonproduction in children with respiratory syncytial, influenza, andparainfluenza virus infections. J. Pediatr. 93:28-32) and does notinduce IFN expression from macrophages in vitro (Roberts, N. J. Jr,Hiscon, J., and D. J. Signs. 1992. The limited role of the interferonsystem in response to respiratory syncytial virus challenge: analysisand comparison to influenza virus challenge. MicrobialPathogenesis.12:409414).

The Applicants have shown that RSV potently induces the cytokinesurpressing inhibitor factor, termed IL-10, by infected humanmacrophages and epithelial cells which directly inhibits IFN production.(Hoffmann, S. P., Rebert, N. A., Panuska, J. R. 1995. RespiratorySyncytial Virus Induction of Lung Cell Expression of Interleukin 10:Implications For Incomplete Immunity. Am. J. of Resp. and Crit. CareMedicine 151:A774). Thus, RSV inhibits expression of anti-viralcytokines by resident lung cells.

However, IFN treatment of RSV infected children does improve theirclinical course suggesting that exogenous IFN may augment intrinsic lungdefenses against this virus (Sung, R. Y. T., Yin, J., Oppenheimer, S.J., Tam, J. S., and J. Lau. 1993. Treatment of respiratory syncytialvirus infection with recombinant interferon alfa-2a. Arch Dis. Child69:440-442).

Applicants therefore determined the effects of TNFα and IFN-β alone, andin combination, on RSV infection and replication in lung epithelialcells as well as the highly RSV permissive CV-1 cell line. Moreover,applicants examined known mechanisms by which TNFα and/or IFN-β restrictvirus replication including inhibition of virus infection, cytostaticeffects on cells, direct lysis of virus infected cells, or inhibition ofprotein synthesis which reflects induction of TNFα/IFN-β responsiveanti-viral genes.

As shown below, applicants have found that IFN-β restricts RSVreplication in human lung epithelial cells, the natural target cells forthis virus. Previously, delivery of ribavirin(1-beta-D-ribofuranosyl-1-1, 2, 4-triazole-3-carboxamide), a syntheticnucleotide that is administered intravenously by small particle aerosolfor 12-20 h a day for approximately three (3) days was essentially theonly anti-viral drug and/or treatment useful in RSV infections. However,ribavirin's clinical efficacy has been marginal and its potentialteratogenic effects have limited its use. Nevertheless, it is used in˜20,000 children per year at a cost of $3,000 per patient.

The present invention relates to the specific delivery of IFN-β to theairway of a host susceptible or suffering from infection of RSV andother RNA respiratory viruses. Recent studies have indicated that INF-βcan be delivered by aerosol to volunteers without causing any harmfuleffects. (Halme, M., Maasilta, P., Mattson, K., Cantell, K. 1994.Pharmacokinetics and Toxicity of Inhaled Human Natural Interferon-Betain Patients With Lung Cancer. Respiration 61:105-107). The invention hasthe ability to restrict RSV without inducing side effects that can beobserved with parenterally administered IFN-β. It also has the potentialto restrict other RNA viruses which might be sensitive to cytokines.

These and other objects of the present invention will be more apparentfrom the discussion below.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of treatingrespiratory syncytial virus (RSV) through the exogenous administrationof recombinant or natural interferon-beta (IFN-β) to lung epithelialcells. Preferably, IFN-β is administered to hosts suffering from RSV asan effective nebulized or aerosolized form.

Along these lines, it has been found through the study of lungepithelial cell lines exposed to RSV that virus replication proceeds ina dose and time dependent manner. In addition, it has been found thatthe administration of exogenous tumor necrosis factor (TNFα) and/orinterferon-beta (INF-β) markedly inhibits RSV in a similar manner.

Specifically, it has been determined that exogenous interferon-beta(INF-β) essentially aborts RSV replication in human lung epithelialcells. Moreover, IFN-β and/or TNFα did not induce cell membrane damage,cause cell lysis, nor inhibit cellular protein synthesis. RSV infectedhuman alveolar macrophages, which produce TNFα, failed to productivelyinfect lung epithelial cells in co-culture. Together these resultsindicate that endogenous TNFα coupled with exogenous IFN-β can overcomethe deficient IFN expression by lung cells to yield restricted RSVreplication. Consequently, the present invention is directed to a methodof treating virus induced respiratory diseases such as RSV through theuse of IFN-β.

In a further aspect, the present invention relates to pharmaceuticalcompositions suitable for use in the treatment of RSV and potentiallyother paramyxovirus via inhalation. The pharmaceutical compositionscontain as the active ingredient, an effective amount of recombinant ornatural IFN-β. In addition, one or more carriers, stabilizers,surfactants, buffers, anti-inflammatory agents, antibiotics, may beincluded in the pharmaceutical compositions in order to enhance theeffectiveness or delivery efficiency of the active agent.

These and other objects and features of the invention will be apparentfrom the following drawings, detailed description of the invention andfrom the claims. It should, however, be understood that the detaileddescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various modifications and changes within the spirit and scope ofthe invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings which are presentedfor the purpose of illustrating the invention and not for the purpose oflimiting same.

FIGS. 1A(i)-1A(iv) show the production of infectious RSV as a functionof viral dose. Lung epithelial and CV-1 cells, as indicated, wereexposed to the indicated doses of RSV and total virus titer (sonicatedcells+supernatants)/10⁶ cells were determined at 48 h p.i. Results shownare mean±SEM, n=5. Virus titer is shown as thousands for BEAS 2B and9HTE cells and millions for A549 and CV-1 cells in this, and subsequentFigures. The increase in virus titer in all cells exposed to RSV>0.1pfu/cell was significant at P<0.01, ANOVA.

FIGS 1B(i)-1B(iv) show the production of RSV as a function of timefollowing infection (MOI=1). Total RSV production/10⁶ cells wasdetermined at the indicated h p.i. Results shown are mean±SEM, n=5. RSVtiter was significantly higher at 48 h in all cell types compared toother time points, P<0.01, Student's t-tests.

FIGS. 2A-2D demonstrate the effects of TNFα on RSV production by lungepithelial and CV-1 cells. Cells were treated with TNFα at the indicateddoses for 16 hours, then exposed to RSV at 1 pfu/cell. After washing,cell monolayers were incubated in fresh media for 48 hours and viraltiter was determined as described in the Examples. Results shown aremean±SEM, n=5. Asterisks indicate significant (P<0.05) differencescompared to untreated controls by Student's t-tests.

FIG. 3A indicates the effects of IFN-β on RSV replication in A549 cells.A549 cells were exposed to IFN-β at the indicated doses for 16 hoursthen infected with RSV (MOI=1). After 48 hours, total RSV production(sonicated cells+supernatants) was determined as described in theExamples. Results shown are mean+SEM, n=4. Doses greater than 2 IU/mlwere significantly (P<0.01) less than controls.

FIG. 3B indicates the effects of TNFα (100 ng/ml) and IFN-β (100 IU/ml)on RSV replication in A549 cells following infection with an MOI=1. A549cells were pretreated (-16 hours), treated simultaneously (0 hours), orpost-treated (4 hours) as indicated with TNFα or IFN-β and the reductionin RSV production was determined compared to untreated cultures. Resultsare mean±SEM, n=4.

FIG. 3C indicates the effects of TNFα and IFN-β alone, and incombination, on RSV replication in A549 cells following infection withan MOI=1. Monolayers were exposed for 16 hours to TNFα (100 ng/ml),IFN-β (2 LU/ml), or both (same concentrations) and total RSV productionwas determined. Results shown are mean+SEM. *P<0.05 comparing TNFα orTNFα/IFN-β to controls.

FIGS. 4A-4D are photomicrographs showing the transmission of RSV betweenalveolar macrophages, 9HTE, and CV-1 cells determined by directimmunofluorescent microscopy as described in the Examples. FIG. 4A, RSVinfected alveolar macrophages at 24 h p.i. were added to uninfectedmonolayers of 9HTE cells. FIG. 4B, RSV infected 9HTE cells were added touninfected alveolar macrophages. FIG. 4C, RSV infected alveolarmacrophages were added to uninfected CV-1 cells. FIG. 4D, RSV infectedCV-1 cells were added to uninfected alveolar macrophages. Alveolarmacrophages (am) were identified by morphology and are indicated byarrows. Co-cultures were fixed and stained for RSV protein expression(yellow-green fluorescence) and Evans blue (red) counterstain. Panels Aand B, magnification×400; FIGS. 4C and 4D, magnification×200.

DETAILED DESCRIPTION OF THE INVENTION

The above and various other objects and advantages of the presentinvention are produced by inhibiting respiratory syncytial virus (RSV)and other respiratory RNA viruses through the administration ofexogenous, recombinant or natural interferon-beta (IFN-β). Preferably,the exogenous IFN-β is administered to the respiratory tract of a hostsusceptible or suffering from infection by RSV and potentially other RNArespiratory viruses by inhalation including the use of effectivenebulized or aerosol doses of IFN-β. Application of exogenous IFN-β vianebulization, coupled with endogenous tumor necrosis factor (TNFα)production by the airway luminal cells in RSV infected patients,interact to restrict RSV replication. This results in an improvedtherapeutic treatment against respiratory virus infections.

In this regard, acute RSV induced lung disease and chronic lungabnormalities are believed to result from replication of virus in airwayepithelial cells yielding syncytia and denudation of the epithelium, aswell as collateral injury of uninfected cells secondary to theinflammatory response. These postulates have not been rigorouslyexamined in human lung epithelial cells nor is it known if these cellspossess intrinsic mechanisms to restrict RSV.

As a result, applicants have employed three human lung epithelial celllines as models (see examples below) to demonstrate that these cells:(i) are permissive to RSV infection and support virus replication in adose/time dependent manner; (ii) respond to TNFα to restrict RSVreplication through an apparent receptor mediated process; (iii) respondto IFN-β alone, or in combination with TNFα, to essentially abort RSVreplication; (iv) respond to pretreatment as well as simultaneoustreatment with TNFα and IFN-β to markedly restrict RSV and; (v) transmitRSV to uninfected alveolar macrophages yet resist transmission of virusfrom RSV infected alveolar macrophages. These results indicate that theexpression of endogenous TNFα or use of exogenous IFN-β have animportant role in restricting RSV replication in human lung epithelialcells.

More particularly, the present invention is directed to the use ofcombining exogenous, nebulized INF-β to the airways coupled withendogenous TNFα, produced by resident alveolar macrophages, to generatetherapeutic benefits in RSV infected individuals. Since RSV replicatesexclusively in luminal respiratory cells, the targets for anti-viraltherapy are organ directed and are narrowly defined. Moreover, since thevolume of epithelial lining fluid (that fluid bathing the luminal airwaycells) is approximately 10 ml in a one (1) year old and 30 ml in a five(5) year old, the amount of nebulized IFN-β necessary to achieve ananti-viral dose is substantially less than that which would be requiredif the drug were administered systemically.

Along these lines, it is noted that while systemic (as opposed tonebulized) administration of IFN-α (and not IFN-β) has been used safelyto treat RSV resulting in some clinical improvement (Sung RYT et al.,Arch. Dis. Child, 69:440-442, 1993), no demonstrable change in virusshedding has been observed. Further, it is noted that this study did notdetermine whether effective levels of IFN were achieved at luminalairway cells. Consequently, these results differ substantially from thepresent invention.

Specifically, as more particularly discussed in the examples below,applicants have determined that IFN-β interacts with appropriatereceptors on airway epithelial cells to restrict RSV replication. Sinceevaluation of the studies set forth in the Examples, applicants havedetermined that IFN-β inhibits RSV replication in the 9HTE (tracheal)and BEAS 2B(bronchiolar) human cell lines. In addition, applicants havealso shown that in six (6) separate human donors, IFN-β inhibits RSVreplication in primary lung epithelial cell explants (unpublishedresults).

These results indicate that human lung epithelial cells expressreceptors which facilitate organ-specific targeted delivery of IFN-β andminimize systemic effects. Furthermore, IFN-β may not induce theprofound immunologic alterations associated with IFNγ that mightadversely alter the immune response to RSV.

As a result, the present invention represents a new and effective way oftreating viral infections in the respiratory tract. Through the use ofIFN-β in nebulized form, therapeutic effects against virus infection areproduced. In the content of the present invention, a therapeuticallyeffective amount of INF-β refers to the amount of INF-β to restrict theproduction of RNA viruses such as RSV in lung epithelial cells.

As used herein, "interferon-β" or "IFN-β" refers to all forms of betainterferon as are known to be active in accepted IFN-β assays, such asby inhibition of encephalomyocarditis virus replication in A549 cells,neutralization by antibodies having immunoreactivity for IFN-β but notIFN-α or IFN-gamma, heat lability, etc . . . Moreover, TNFα, as employedherein, refers, in general, to the various forms of TNF-alpha whichexhibit one or more biologic properties of tumor necrosis such as tumorcell lysis, inhibition of infectious agents, and are neutralized byantibodies to TNF-alpha (α) but not antibodies to TNF-beta (lymphotoxin)or other cytokines.

In addition, the IFN-β utilized in this invention is that of recombinantor natural type. Specifically, IFN-β produced in recombinant cellculture, from natural isolates or by stable untransformed cell lines aresatisfactory for use herein. Suitable IFN-β, includes those availablefrom Toray Pharmaceuticals, Hoffman La Roche and others. Optionally,TNFα may also be included in the method and composition of the presentinvention. The TNFα utilized includes the products of recombinant oruntransformed cell culture. Suitable TNFα include those available fromGenentech. In practice of the invention, the dosage of IFN-β and/or TNFαwill vary as it is understood by one skilled in the art. Severalvariables will be taken into account in determining the appropriateproperties of IFN-β or TNFα to be used, the concentration of INF-βand/or TNFα in the therapeutic compositions and the dosages to beadministered. These include, the clinical conditions of the patients andothers. Consequently, as it is understood by those of skill in the art,dosages and treatment regimens will typically be modified according tothe attendant circumstances and medical conditions.

For the purposes of the present invention, lyophilized human IFN-β isprovided at 3×10⁶ IU/vial (calibrated by NIH international standard) byToray Industries, Inc. of Tokyo, Japan. In addition to the purifiedhuman INF-β protein, the lyophilized preparation control contains 9mg/vial of human serum albumin and 1 mg/vial of D-lactose. Forutilization, the lyophilized human INF-β preparation is dissolved in apharmaceutically accepted vehicle such as sterile distilled water and/orsterile physiological saline. In addition, one or more carriers,stabilizers, surfactants, buffers, anti-inflammatory agents,antibiotics, etc., may be included in the IFN-β preparation in order toenhance the effectiveness of the IFN-β active agent. The IFN-βpreparations are provided as sterile aqueous solutions.

Preferably, the IFN-β is administered by inhalation over several minuteswith use of a nebulizer or the like. As briefly indicated, dosage andfrequency of inhalation may vary, depending on the patient's symptoms orconditions.

A wide variety of nebulizers can be utilized to deliver the compositionof the present invention to the lung epithelial cells. As definedherein, a nebulizer includes all means of delivering the IFN-βcompositions of the present invention in a fine spray or mist Fromliquid. The size of the particle produced will depend upon the method ofnebulization utilized. The fine spray or mist may be produced by passingair through a liquid or by vibrating a liquid at a high frequency sothat the particles produced are extremely small. In addition, thecomposition can be propelled by a pressure differential created by therelease of a pressurized propellant or by a stream of air drawn throughor created by a mechanical device.

The method and composition of this invention are useful in preventing ortreating active infections of RNA viruses such as RSV. Other specificviruses include the paramyxoviruses such as parainfluenza viruses I, II,III, IV, mumps, and avian parainfluenza virus type 1-6.

Further, the method and composition of the present invention is usefulin treating RNA virus infection, such as RSV, in mammals, including man,bovine, and primates. Since these viruses are conveniently transmittedthrough the respiratory tract, use of the nebulized or aerosolizedcompositions of the present invention is deemed to be an effective routeof administration. This is particularly true since INF-β can beadministered as a nebulized pharmaceutical agent without toxicity.

EXAMPLES

The following examples serve to illustrate the present invention and arenot intended to limit the scope of this invention

MATERIALS AND METHODS

Cell lines

A549, CV-1, and U937 cell lines were obtained from the American TissueCulture Collection (ATCC CRL #185, 70, 1593, respectively) and werepropagated in DMEM (JRH Biosciences, Lenexa, Kans.), EMEM (Sigma, St.Louis, Mo.), or RPMI 1640 (Gibco BRL, Life Technologies, Grand Island,N.Y.), respectively, supplemented with 10% (v/v) fetal bovine serum(Hyclone, Logan UT), 1 mM non-essential amino acids, 1 mM sodiumpyruvate, 2 mM L-glutamine, 100 μg/ml penicillin, 100 μg/ml streptomycinand 0.25 μg/ml amphotericin B (all from Sigma, St. Louis, Mo.) (DMEM,EMEM or RPMI culture media). 9HTE cells were kindly provided by Dr. D.C. Gruenert (Cardiovascular and Cancer Research Institute, University ofCalifornia, San Francisco Calif.) and were propagated in MEM culturemedia as described (Gruenert, D. C., Basbaum, C. B., Welsh, M. J., Li,M., Finkbeiner. W. E., and J. A. Nadel. 1988. Characterization of humantracheal epithelial cells transformed by an origin defective simianvirus 40 Proc. Natl. Acad. Sci. 85:5951-5955). BEAS 2B cells were kindlyprovided by Dr. C. C. Harris (Laboratory of Human Carcinogenesis,National Cancer Institute, Bethesda, Md.) and were propagated in LHC-8media (BioFluids, Rockville Md.) supplemented with 100 μg/ml penicillin,100 μg/ml streptomycin and 0.25 μg/ml amphotericin B as described(Reddel, R. R., Ke, Y., Gerwin, B. l., McMenamin, M. G., Lechner, J. F.,Su, R. T., Brash, D. E., Park, J. B., Rhim, J. S., and C. C. Harris.1988. Transformation of human bronchial epithelial cells by infectionwith SV40 or adenovirus-12 SV40 hybrid virus, or transfection viastrontium phosphate coprecipitation with a plasmid containing SV40 earlyregion genes. Can. Res. 48:1904-1909). All cell lines were passagedtwice a week.

Preparation of alveolar macrophaces

Fourteen normal, non-smoking donors without respiratory disease orsymptoms of viral infection within the preceding four weeks underwentbronchoscopy and bronchoalveolar lavage exactly as applicants havedescribed previously (Panuska, J. R., Midulla, F., Cirino, N. M.,Villani, A., Gilbert, I. A., McFadden, E. R., and Y. T. Huang. 1990.Virus-induced alterations in macrophage production of tumor necrosisfactor and prostaglandin E₂. Am. J. Physiol (Lung Cell Mol Physiol)259:L396-L402; Panuska, J. R., Cirino, N. M., Midulla, F., Despot, J.E., McFadden, E. R., and Y. T. Huang. 1990. Productive infection ofisolated human alveolar macrophages by respiratory syncytial virus. J.Clin. Invest 86:113-119). All studies were approved by the institutionalreview board of University Hospitals of Case Western Reserve Universityand informed written consent was obtained from all donors. Alveolarmacrophages were purified by adherence to plastic tissue culture dishesas previously described (Panuska, J. R., Midulla, F., Cirino, N. M.,Villani, A., Gilbert, I. A., McFadden, E. R., and Y. T. Huang. 1990.Virus-induced alterations in macrophage production of tumor necrosisfactor and prostaglandin E₂. Am. J. Physiol (Lung Cell Mol Physiol)259:L396-L402; Cirino, N. M., Panuska, J. R., Villani, A., Taraf, H.,Rebert, N. A., Merolla, R., Tsivitse, P., and I. A. Gilbert. 1993.Restricted replication of respiratory syncytial virus in human alveolarmacrophages. J. Gen. Virol. 74:1527-1537; Panuska, J. R., Cirino, N. M.,Midulla, F., Despot, J. E., McFadden, E. R., and Y. T. Huang. 1990.Productive infection of isolated human alveolar macrophages byrespiratory syncytial virus. J. Clin. Invest 86:113-119) for 1 hour at37° C. in 5% CO₂ in RPMI 1640 containing 10% (v/v) fetal bovine serumand the antibiotic supplements listed above.

RSV propagation, infection and replication

RSV stocks were prepared in CV-1 cells as previously described (Cirino,N. M., Panuska, J. R., Villani, A., Taraf, H., Rebert, N. A., Merolla,R., Tsivitse, P., and I. A. Gilbert. 1993. Restricted replication ofrespiratory syncytial virus in human alveolar macrophages. J. Gen.Virol. 74:1527-1537; Panuska, J. R., Cirino, N. M., Midulla, F., Despot,J. E., McFadden, E. R., and Y. T. Huang. 1990. Productive infection ofisolated human alveolar macrophages by respiratory syncytial virus. J.Clin. Invest 86:113-1193) and stored at -70° C. until used. Cell lineswere infected with RSV at the multiplicity of infection (MOI) listed inthe text while alveolar macrophages were infected at an MOI of 3pfu/cell. Adherent cell monolayers were exposed to virus at 37° C. in 5%CO₂ for 2 hours then virus inocula were removed by two washes withculture media. Cultures were incubated for the times listed in the textat 37° C. in 5% CO₂.

Percentage of RSV infected cells were determined by directimmunofluorescent staining with monoclonal antibodies (Mab) directedagainst RSV surface glycoproteins (gift of Bartels Immunodiagnostics,Bellevue, Wash.) using methods previously described (Panuska, J. R.,Hertz, M. l., Taraf, H., Villani, A., and N. M. Cirino. 1992.Respiratory syncytial virus infection of alveolar macrophages in adulttransplant patients. Am. Rev. Respir. Dis. 145:934939; [Midulla, F.,Villani, A., Panuska, J. R., Dab, l., Kolls, J. K., and R. Merolla.1993. Respiratory syncytial virus lung infection in infants:Immunoregulatory role of infected alveolar macrophages. J. Infect Dis.168:1515-1519.] Cirino, N. M., Panuska, J. R., Villani, A., Taraf, H.,Rebert, N. A., Merolla, R., Tsivitse, P., and I. A. Gilbert. 1993.Restricted replication of respiratory syncytial virus in human alveolarmacrophages. J. Gen. Virol. 74:1527-1537; Panuska, J. R., Cirino, N. M.,Midulla, F., Despot, J. E., McFadden, E. R., and Y. T. Huang. 1990.Productive infection of isolated human alveolar macrophages byrespiratory syncytial virus. J. Clin. Invest 86:113-119).

Briefly, epithelial cells were plated on eight well LabTech™ slides(Nunc Inc., Naperville, Ill.) at the doses and times described in thetext, then fixed with 4% (wt/vol) paraformaldehyde in a buffercontaining 25 mM HEPES, 60 mM PIPES, pH 6.9, 5 mM MgCl₂ and 1.5 mM GTP.After 30 min, slides were treated with 0.1% Triton X100 in 10 mMphosphate, pH 7.4, 150 mM NaCl (PBS) for 90 seconds then washed fourtimes in PBS containing 1 mg/ml bovine serum albumin (PBS/BSA). Slideswere then reacted for 1 hour at room temperature with anti-RSV Mabcoupled to fluorescein isothiocyanate. Slides then were washed 3 timeswith PBS/BSA and overlaid with PBS 10% (v/v) glycerol. Slides wereviewed with a Nikon Diaphot microscope under bright light andepifluorescent microscopy to enumerate total and fluorescent positivecells, respectively. The percentage of fluorescent positive/total cellswere determined on at least 300 cells per condition.

Virus replication was determined in aliquots of sonicated cells culturesby adding serial two-fold dilutions in triplicate to monolayers of CV-1cells grown on 96 well tissue culture plates exactly as describedpreviously (Cirino, N. M., Panuska, J. R., Villani, A., Taraf, H.,Rebert, N. A., Merolla, R., Tsivitse, P., and I. A. Gilbert. 1993.Restricted replication of respiratory syncytial virus in human alveolarmacrophages. J. Gen. Virol. 74:1527-1537; Panuska, J. R., Cirino, N. M.,Midulla, F., Despot, J. E., McFadden, E. R., and Y. T. Huang. 1990.Productive infection of isolated human alveolar macrophages byrespiratory syncytial virus. J. Clin. Invest 86:113-119)). Viral titeris expressed as plaque forming units/10⁶ cells to correct fordifferences in cell size/number at equivalent levels of monolayerconfluences.

Cytokine Effects on RSV Replication

A549, BEAS 2B, 9HTE and CV-1 cell lines were treated with trypsin(Sigma, St. Louis, Mo.), counted by trypan blue exclusion, and plated ata concentration of either 3.5×10⁵ cells/ml (A549, 9HTE, CV-1), or5.0×10⁵ cells/ml. (BEAS 2B, because of their smaller size and lowerprotein content per cell). A549, BEAS 2B, 9HTE and CV-1 cell monolayerswere incubated for the times indicated in the text at 37° C. in 5% CO₂with recombinant TNFα (a gift of Genentech, San Francisco Calif.) and/orhuman IFN-β (a kind gift of Dr. Jun Utsumi, Toray Industries Inc. Tokyo,Japan) at the doses listed in the text. Cells were then infected for 2hours with RSV at an MOI of 1, washed twice in fresh culture media, andincubated for 48 hours. The epithelial monolayers were harvested byscraping and ice-cold aliquots were sonicated twice at maximal output inan Artek Sonic Dismembrator prior to titration for infectious virus asdescribed above.

TNFα Receptor Assays

[¹²⁵ I]-TNFα (specific activity 42 μCi/μg) was purchased from AmershamCorp. (Arlington Heights, Ill.). A549, 9HTE, BEAS 2B, and CV-1 weregrown as monolayers in tissue culture plates and washed twice PBS/BSA.U937 cells were treated with phorbol myristeric acetate for 24 h asdescribed previously (Villani, A., Cirino, N. M., Baldi, E., Kester, M.,McFadden, E. R., J. R. Panuska. 1991. Respiratory syncytial virusinfection of human mononuclear phagocytes stimulates synthesis ofplatelet-activating factor. J. Biol Chem. 266:5472-5479), washed twicewith PBS/BSA, then used in assays. [¹²⁵ I]-TNFα was added to duplicatewells and incubated at 4° C. for 1 hour. Duplicate wells containing 100fold molar excess of recombinant TNFα were analyzed in parallel. Unboundradioactivity was aspirated and cell monolayers were washed twice inice-cold PBS/BSA. Monolayers were lysed with 0.5% (w/v) NP-40 andcounted in a Micromedic Apex® gamma counter. Parallel wells containingmonolayers of each cell type were lysed with distilled water and proteinconcentration determined utilizing a dye protein assay (Bio-RadLaboratories, Melville, N.Y.). Specific binding was expressed as countsper minute per mg of cellular protein.

Cellular expression of the 55 and 75 kDa TNFα receptors were determinedby enzyme linked immunoabsorbent assays using minor modifications ofpreviously described methods (Higuchi, M., and B. B. Aggarwal. 1992.Microtiter plate radioreceptor assay for tumor necrosis factor and itsreceptors in large numbers of samples. Analytical Biochem. 204:53-58).Briefly, lung epithelial, CV-1, or U937 cells were washed×3 in PBS, twofold serially diluted in PBS, then fixed in 100% ice-cold methanol for 5minutes. Fixed cells were then treated for 5 minutes in 6% H₂ O₂ in 94%methanol. Fixed cells were washed×2 in PBS/BSA and incubated in theabsence or presence of Mab to the 55 and/or 75 kDa TNFα receptor(Genzyme, Cambridge, Mass.) at 20 μg/ml in PBS for 1 hour at 4° C. Cellswere washed×2 with PBS/BSA then reacted with biotin labeled goatanti-mouse antibodies at 0.1 μg/ml (Kirkegaard and Perry, Gaithersburgh,Md.) and incubated on ice for 30 minutes. Cells were then washed×2 inPBS/BSA and reacted with 0.1 μg/ml streptavidin peroxidase (Kirkegaardand Perry, Gaithersburgh, Md.) and incubated on ice for 30 minutes.Cells were then washed×2 in PBS/BSA and centrifuged at 2000 rpm for 10minutes. Cell pellets were resuspended in 250 μl H₂ O₂ and dispensed in50 μl aliquots to wells of a 96 well tissue culture dish. 150 μl ofdiaminobenzidine HCI (25 mg/ml) was added to each well and incubated for15 minutes and absorbance at 450 nm was determined with a microtiterabsorbance meter (E MAX™ Molecular Devices, Menlo Park, Calif.).Controls included samples without cells; lung epithelial cells reactedwith irrelevant isotype control Mabs and the secondary antibodies; cellsreacted with the secondary antibodies alone; or cells reacted in theabsence or primary or secondary antibodies but exposed todiaminobenzidine HCI alone. In each case, absorbance at 450 nm waswithin 10% of media controls. Standard curves were generated with U937cells shown previously to express the 55 and 75 kDa TNFα receptors(Higuchi, M., and B. B. Aggarwal. 1992. Microtiter plate radioreceptorassay for tumor necrosis factor and its receptors in large numbers ofsamples. Analytical Biochem. 204:53-58). Absorbance was determined atcell concentrations of 0, 0.2, 0.4, 1.0, 2.0 and 3.0×10⁶ cells/ml ontriplicate samples. Results were analyzed by linear regression analysis.

Cell Viability and Protein Assays

The effects of TNFα and IFN-β on cell viability were determined bytrypan blue exclusion and enumeration of the percentage of cells thatexcluded the dye by light microscopy. Membrane damage of epithelialcells were determined by reacting monolayers with acridine orange andethidium bromide (each at 1 μg/ml in PBS) and determining the percentageof membrane damaged cells by fluorescent microscopy as previouslydescribed (Cirino, N. M., Panuska, J. R., Villani, A., Taraf, H.,Rebert, N. A., Merolla, R., Tsivitse, P., and l. A. Gilbert. 1993.Restricted replication of respiratory syncytial virus in human alveolarmacrophages. J. Gen. Virol. 74:1527-1537; Panuska, J. R., Cirino, N. M.,Midulla, F., Despot, J. E., McFadden, E. R., and Y. T. Huang. 1990.Productive infection of isolated human alveolar macrophages byrespiratory syncytial virus. J. Clin. Invest 86:113-119). Acridineorange stains the nuclei of viable cells green while ethidium bromidestains the nuclei of nonviable cells orange. The effects of TNFα andIFN-β on cell growth were determined by extensively washing monolayerswith PBS, lysing the monolayers with distilled water, and two cycles offreeze/thaw. Cellular protein concentration was then determined asdescribed above as a function of time following treatment with thesecytokines or media controls. Cellular proteins were analyzed by sodiumdodecyl sulfate (SDS) electrophoresis on 10% (w/v) polyacrylamide gelswith Coomassie blue staining as previously described (Panuska, J. R.,Fukui, K., and C. W. Parker. 1988. Secreted proteins of human monocytes.Biochem. J. 249:501-511).

Transmission of RSV between Epithelial, CV-1 cells and AlveolarMacrophages.

Epithelial cells were infected with RSV at an MOI=1 then harvested at 24hours post-infection and added to monolayers of uninfected alveolarmacrophages. In parallel, alveolar macrophages were infected with RSV atan MOI=3 then harvested at 24 h post-infection and added to monolayersof uninfected epithelial cells. In some experiments, RSV infectedalveolar macrophages were mixed with 500 U/ml of a neutralizing antibodyto TNFα (Amersham Corp., Arlington Heights, Ill.) prior to addition toepithelial monolayers. Co-cultures were overlaid with 0.5% (w/v) agarosein culture media and incubated for 48 hours at 37° C. in 5% CO₂.Co-cultures were then fixed in methanol/acetone as previously described(Panuska, J. R., Hertz, M. I., Taraf, H., Villani, A., and N. M. Cirino.1992. Respiratory syncytial virus infection of alveolar macrophages inadult transplant patients. Am. Rev. Respir. Dis. 145:934939; Cirino, N.M., Panuska, J. R., Villani, A., Taraf, H., Rebert, N. A., Merolla, R.,Tsivitse, P., and I. A. Gilbert. 1993. Restricted replication ofrespiratory syncytial virus in human alveolar macrophages. J. Gen.Virol. 74:1527-1537) and stained for RSV protein expression by directimmunofluorescence as described above. Cell types were discriminated bymorphology.

Statistical Analysis

All results shown are mean±standard deviation (SD) or standard error ofthe mean (SEM) as indicated in the text. The effects of dose weredetermined by analysis of variance (ANOVA) and comparisons between meanswere determined by Student's t-tests. Multiple t-test analyses werecorrected by the Bonferroni method. Probability (P) values less than0.05 were considered significant.

RESULTS

RSV replication in BEAS 2B, A549, and 9HTE epithelial cells

BEAS 2B, A549, 9HTE and CV-1 cells exposed to RSV, then extensivelywashed to remove virus inocula, demonstrated a significant (P<0.01,ANOVA) virus dose dependent increase in virus production at 48 h p.i.,FIG. 1A. Virus production in sonicated cells and supernatants weremaximal in all cell lines at 48 h p.i. (P<0.01, Student's t-tests), FIG.1B. Accumulation of infectious RSV was decreased at 72 and 96 h p.i.probably due to lysis of epithelial cell monolayers and instability ofvirus at 37° C. as previously reported (Mcintosh, K. and R. M. Chanock.1990. Respiratory syncytial virus. In B. N. Field, D. M. Knipe et al.,editors. Virology, Second Edition, Raven Press, Ltd., New York, Chapter38, 1045-1072; Cirino, N. M., Panuska, J. R., Villani, A., Taraf, H.,Rebert, N. A., Merolla, R., Tsivitse, P., and I. A. Gilbert. 1993.Restricted replication of respiratory syncytial virus in human alveolarmacrophages. J. Gen. Virol. 74:1527-1537). Virus replication per 10⁶cells at 48 h p.i. was highest in CV-1 (˜4×10⁶)>A549 (˜1.8×10⁶)>9HTE(˜0.1×10⁶)>BEAS 2B cells (˜0.1×10⁶). All cell lines released infectiousvirus but viral titer in cell-free supernatants were less than 20% oftotal cellular virus at all time points (not shown). These resultssuggested that the site of origin, transformation with SV-40 (BEAS 2Band 9HTE), or the state of differentiation of these lung epithelialcells determined their capacity to replicate RSV.

Effects of TNFα and IFN-β on RSV replication in lung epithelial cells

RSV induces alveolar macrophages to produce TNFα in vivo (Midulla, F.,Villani, A., Panuska, J. R., Dab, I., Kolls, J. K., and R. Merolla.1993. Respiratory syncytial virus lung infection in infants:Immunoregulatory role of infected alveolar macrophages. J. Infect Dis.168:1515-1519; Hayes, P. J., Scok, R., and J. Wheeler. 1994. In vivoproduction of tumor necrosis factor α and interleukin-6 in BALB/c miceinoculated intranasally with a high dose of respiratory syncytial virus.J. Med. Virol 42:323-329) which can function as a potent anti-viralcytokine (Cirino, N. M., Panuska, J. R., Villani, A., Taraf, H., Rebert,N. A., Merolla, R., Tsivitse, P., and l. A. Gilbert. 1993. Restrictedreplication of respiratory syncytial virus in human alveolarmacrophages. J. Gen. Virol. 74:1527-1537; Wong, G. H. W., Tartaglia, L.A., Lee, M. S., and D. V. Goeddel.1992. Antiviral activity of tumornecrosis factor (TNF) is signaled through the 55-kDa receptor, type 1TNF. J. Immunology 149:3350-3353; Wong, G. H. W., Kamb, A., and D. V.Goeddel.1993. Antiviral properties of TNF. In B. Beutler, editor. TumorNecrosis Factors: The Molecules and Their Emerging Role in Medicine.Raven Press. Ltd, New York, 371-381). Pretreatment of lung and CV-1cells with recombinant TNFα (rTNFα) yielded a significant (P<0.05,ANOVA) dose dependent inhibition of RSV replication in BEAS 2B, A549,and 9HTE cells, but not CV-1 cells, compared to media controls, FIG. 2.CV-1 cells pretreated with rTNFα at doses as high as 5000 μg/mlreplicated RSV as efficiently as untreated cells, consistent with priorwork (Cirino, N. M., Panuska, J. R., Villani, A., Taraf, H., Rebert, N.A., Merolla, R., Tsivitse, P., and l. A. Gilbert. 1993. Restrictedreplication of respiratory syncytial virus in human alveolarmacrophages. J. Gen. Virol. 74:1527-1537).

TNFα and IFN-β can interact to restrict replication of some viruses.Pretreatment of lung cells with IFN-β (100 IU/ml) for 16 hours markedlyrestricted RSV replication, Table 1.

                  TABLE 1                                                         ______________________________________                                        Effects of IFN-β (100 IU/ml) on lung epithelial and                        CV-1 cell replication of RSV.                                                   CELL TYPE % INHIBITION OF RSV REPLICATION*                                ______________________________________                                        CV-1      83.2 ± 12.2                                                        BEAS 2B 92.2 ± 4.5                                                         9HTE 77.8 ± 13.1                                                           A549 96.6 ± 8.6                                                          ______________________________________                                         *Results shown are mean ± SD of triplicate samples each twofold            serially diluted to provide replicates of six. Results are from a single      experiment, performed in parallel, representative of two separate             experiments.                                                             

In contrast to TNFα, IFN-β potently inhibited RSV replication in CV-1cells indicating these cytokines can activate separate anti-viralpathways. The effects of IFN-β dose were examined with A549 lungepithelial cells. Pretreatment with IFN-β for 16 h prior to infectionwith RSV (MOI=1) demonstrated a dose dependent inhibition of RSVreplication in A549 cells yielding essentially complete inhibition ofRSV replication at doses>20 IU/ml, FIG. 3A. IFN-β doses of 20 IU/ml wereas potent in inhibiting RSV replication as was TNFα (compare with FIG.2). To investigate if pretreatment with TNFα and/or IFN-β was necessaryto inhibit RSV replication, A549 cells were pretreated with thesecytokines for 16 hours, simultaneously treated at the time of RSVexposure, or treated at 4 h p.i. Simultaneous treatment decreased RSVreplication to a similar extent as pretreated cells, however addition ofTNFα or IFN-β at 4 h p.i. was significantly (P<0.05) less effective,FIG. 3B. To determine if TNFα and IFN-β interacted to inhibit RSV, A549cells were pretreated with submaximal inhibitory doses of TNFα (100μg/ml) and IFN-β (2 IU/ml) separately, and in combination. As shown inFIG. 3C. TNFα and IFN-β interacted to inhibit RSV replication. Thisinteraction appeared additive, and not synergistic, when analyzed byisobolographic analysis (not shown).

TNFα Receptor Expression

The TNFα mediated dose (FIG. 2) and time (FIG. 3) dependent inhibitionof RSV replication in lung epithelial, but not CV-1 cells, suggestedthat these cells might differentially express TNFα receptors that couldtransduce the signal resulting in restricted viral replication. [¹²⁵I]-TNFα binding studies of adherent cell monolayers and U937 cells, as apositive control (Higuchi, M., and B. B. Aggarwal. 1992. Microtiterplate radioreceptor assay for tumor necrosis factor and its receptors inlarge numbers of samples. Analytical Biochem. 204:53-58), were performedin the absence or presence of a 100 fold excess of rTNFα. Resultsindicated that all cell types specifically bound TNFα although lessbinding was observed with CV-1 cells per mg cellular protein than wereseen with BEAS 2B and 9HTE cells, Table 2.

                  TABLE 2                                                         ______________________________________                                        TNFα receptors on lung epithelial and CV-1 cells                                    [.sup.125 I]TNF bound                                                                     55 kDa receptor                                                                          75 kDa receptor                               (cpm/mg cellular (absorbance/ (absorbance/                                   Cell Type protein) 10.sup.6 cells) 10.sup.6 cells)                          ______________________________________                                        CV-1    3,072 ± 1,055                                                                          0.17 ± 0.02                                                                             0.13 ± 0.01                                 BEAS 2B 54,596 ± 7,892*  0.35 ± 0.05* 0.17 ± 0.05                    9HTE 11,461 ± 4,991* 0.25 ± 0.02 0.13 ± 0.06                         A549 4,721 ± 1,756  0.57 ± 0.14* 0.22 ± 0.08                         U937 15,494 ± 1,523* 0.26 ± 0.02  0.33 ± 0.02*                     ______________________________________                                         Results shown are mean ± SD, n = 3 for [.sup.125 I]TNFα binding.     TNFα receptor expression for the 55 and 75 kDa subtypes was             determined by ELISA and results shown were determined by linear regressio     analysis of absorbance versus cell number as described in Methods.            *P, 0.05 compared to CV1 cells (Student's ttests).                       

Consistent with these results, all cell types expressed detectableproteins for the 55 and 75 kDa TNFα receptors assessed by ELISA withMabs to these receptor subtypes, Table 2. U937 cells express both the 55and 75 kDa TNFα receptors (Higuchi, M., and B. B. Aggarwal. 1992.Microtiter plate radioreceptor assay for tumor necrosis factor and itsreceptors in large numbers of samples. Analytical Biochem. 204:53-58)and were used as a positive control. Omission of either the primary Mabsor secondary antibodies yielded absorbance values that did not differ bygreater than 10% from media controls. Although TNFα binding and proteinexpression did not show strict concordance, this could result fromdifferences in receptor affinity, steric availability on cell membranes,or receptor turnover. Nevertheless, expression of the 55 and 75 kDa TNFαreceptors did not appear to account for the differential effects of TNFαon RSV replication in lung epithelial versus CV-1 cells.

Effects of TNFα on RSV Infection, Cell Growth, and Viability ofEpithelial Cells

TNFα can restrict virus replication by interfering with infection,inhibiting cell growth, lysis of virus infected cells, or induction ofother anti-viral pathways (Cirino, N. M., Panuska, J. R., Villani, A.,Taraf, H., Rebert, N. A., Merolla, R., Tsivitse, P., and l. A. Gilbert.1993. Restricted replication of respiratory syncytial virus in humanalveolar macrophages. J. Gen. Virol. 74:1527-1537; Wong, G. H. W.,Tartaglia, L. A., Lee, M. S., and D. V. Goeddel.1992. Antiviral activityof tumor necrosis factor (TNF) is signaled through the 55-kDa receptor,type 1 TNF. J. Immunology 149:3350-3353; Wong, G. H. W., Kamb, A., andD. V. Goeddel.1993. Antiviral properties of TNF. In B. Beutler, editor.Tumor Necrosis Factors: The Molecules and Their Emerging Role inMedicine. Raven Press. Ltd, New York, 371-381). These possiblemechanisms were examined sequentially by applicants.

All lung epithelial cells, when pretreated with rTNFα (1000 ng/ml) priorto RSV infection (MOI=0.1), demonstrated a small 30±8%, but significant(n=4, P<0.05), reduction in infected cells at 48 h p.i. determined bydirect immunofluorescence compared to untreated cells. This was notobserved with lung cells infected with a 10 fold higher RSV dose (MOI=1)(93+4 vs. 96+6% infected cells in rTNFα pretreated vs. media controls,respectively, n=4, P=NS). Thus, sufficient RSV dose overcame theanti-infective effects of TNFα and suggested that TNFα could alsointerfere with virus replication at a step distal to initial infection.

TNFα did not significantly reduce cell growth or viability of BEAS 2B,A549 and 9HTE cells. All lung epithelial cells pretreated for 16 h withrTNFα (1000 ng/ml) demonstrated a 3.1±0.8 fold increase in cellularprotein concentration and a 2.9±0.9 fold increase in viable cell numberat 48 h post-treatment which differed by less than 20% compared toepithelial cells treated with culture media alone (n=3, P=NS). Cellmembrane integrity (acridine orange/ethidium bromide staining) orviability (trypan blue exclusion) in TNFα pretreated lung epithelialcells that were mock or RSV infected were indistinguishable from mediacontrols assessed at 24 h p.i. Under all conditions, with eachepithelial cell type, the percentage of cells with intact membranes andviable was greater than 86% (n=2).

Finally, mock or RSV infected A549 and 9HTE cells pretreated for 16hours with IFN-β, TNFα, or both, demonstrated essentially equivalentexpression of cellular proteins compared to uninfected controls whenlysates from equal cell numbers were analyzed by SDS polyacrylamideelectrophoresis (not shown).

Transmission of RSV between epithelial cells and human alveolarmacrophapes.

TNFα restricts RSV replication in alveolar macrophages and inhibits RSVinfection of lung epithelial cells at low MOI (see above). To examine ifTNFα, endogenously expressed by RSV infected alveolar macrophages,effected RSV transmission between these cells and lung epithelial cells,co-culture experiments were performed. Transmission of RSV from infectedcells (24 hours p.i.) to uninfected cells were determined after 24 hoursof co-culture under 0.5% agarose to prevent fluid phase spread of virus.RSV infection was assessed by direct immunofluorescence and cell typeswere discriminated by morphology. Infected 9HTE cells transmitted RSV touninfected alveolar macrophages (FIG. 4) In contrast, RSV infectedalveolar macrophages clearly adhered to epithelial monolayers but didnot transmit virus to 9HTE cells, (FIG. 4B), A549 or BEAS 2B cells (notshown). Although alveolar macrophages did not transmit RSV to these lungepithelial cells, they were competent to transmit virus to the RSVpermissive CV-1 cell line (FIG. 4C). RSV infected CV-1 cells transmittedRSV to uninfected alveolar macrophages similar to lung epithelial cells(FIG. 4D). These results are consistent with results applicants havepreviously described (Cirino, N. M., Panuska, J. R., Villani, A., Taraf,H., Rebert, N. A., Merolla, R., Tsivitse, P., and l. A. Gilbert. 1993.Restricted replication of respiratory syncytial virus in human alveolarmacrophages. J. Gen. Virol. 74:1527-1537).

In fourteen separate donors, RSV was transmitted solely from epithelialcells to uninfected alveolar macrophages and not from RSV infectedalveolar macrophages to uninfected epithelial cell lines. Transmissionof RSV from infected alveolar macrophages to 9HTE cells was not alteredby addition of anti-TNFα or anti-IFN-β (1000 neutralizing units/ml) toco-cultures. However, applicants interpret these results cautiouslybecause it is possible that these cytokines interact with theirreceptors through sequestered spaces or prior to capture by theneutralizing antibodies.

DISCUSSION

As indicated above, acute RSV induced lung disease and chronic lungabnormalities are thought to result from replication of the virus inairway epithelial cells yielding syncytia and denudation of theepithelium, as well as collateral injury of uninfected cells secondaryto the inflammatory response. These postulates have not been rigorouslyexamined in vitro in human lung epithelial cells nor is it known ifthese cells possess intrinsic mechanisms to restrict RSV.

As a result, applicants here employ three human lung epithelial cells asmodels to demonstrate that these cells: (i) were permissive to RSVinfection and supported virus replication in a dose/time dependentmanner; (ii) responded to TNFα to restrict RSV replication through anapparent receptor mediated process; (iii) responded to IFN-β alone, orin combination with TNFα, to essentially abort RSV replication; (iv)responded to pretreatment as well as simultaneous treatment with TNFαand IFN-β to markedly restrict RSV and; (v) transmitted RSV touninfected alveolar macrophages yet resisted transmission of virus fromRSV infected alveolar macrophages. Thus, it has been found thatexpression of endogenous TNFα or use of exogenous IFN-β have a role inrestricting RSV replication in human lung epithelial cells.

Lung epithelial cells supported RSV replication less efficiently thanCV-1 cells. Applicants have recently discovered that lung epithelialcell lines, and primary lung epithelial cells, express constitively theenzyme 2', 5' oligoadenylate (2', 5'A) synthetase dependent RNase Lwhich degrades viral m RNA transcripts and restricts viral replication.The 2', 5'A dependent RNase L is induced by IFN-β and may serve as thecritical molecular pathway to restrict RSV replication in lungepithelium. (Panuska, J. R., Rebert, N. A., Hoffmann, S. I.Anti-Respiratory Syncytial Virus Pathways in Cytokine Treated Human LungCells. Am. J. of Resp. and Crit. Care Medicine 151:A122). These resultssuggest that the differentiated phenotype of lung epithelial cells mayregulate their capacity to replicate RSV. A549 cells, of alveolarorigin, efficiently replicated RSV and may provide a cellular model forRSV induced pneumonia which primarily effects alveolar cells. 9HTE andBEAS 2B cells derived from tracheal and bronchiolar origin, alsoreplicated RSV, albeit less efficiently than A549 cells, but again mayprovide a cellular model for RSV induced conducting airway andbronchiolar inflammation. The results presented here do not permitapplicants to determine if differentiated phenotype, transformation withSV-40, anatomical origin, or species derivation (human lung vs. monkeykidney cell) serve as the primary determinants controlling theefficiency of RSV replication (Gruenert, D. C., Basbaum, C. B., Welsh,M. J., Li, M., Finkbeiner. W. E., and J. A. Nadel. 1988.Characterization of human tracheal epithelial cells transformed by anorigin defective simian virus 40 Proc. Natl. Acad. Sci. 85:5951-5955;Reddel, R. R., Ke, Y., Gerwin, B. l., McMenamin, M. G., Lechner, J. F.,Su, R. T., Brash, D. E., Park, J. B., Rhim, J. S., and C. C. Harris.1988. Transformation of human bronchial epithelial cells by infectionwith SV40 or adenovirus-12 SV40 hybrid virus, or transfection viastrontium phosphate coprecipitation with a plasmid containing SV40 earlyregion genes. Can. Res. 48:1904-1909; Standiford, T. J., Kunkel, S. L.,Basha, M. A., Chensue, S. W., Lynch, J. P., Toews, G. B., Westwick, J.,and R. M. Strieter. 1990. Interleukin-8 gene expression by a pulmonaryepithelial cell line. J. Clin. Invest 86:1945-1953). Further studiescould address these possibilities.

The state of differentiation did appear to differ between lungepithelial and CV-1 cells. Lung epithelial cells treated with rTNFαmarkedly restricted RSV replication whereas CV1 cells were unresponsive.This did not simply reflect a lack of expression of the 55 or 75 kDaTNFα receptors which may transduce the anti-viral signal(s) (Wong, G. H.W., Tartaglia, L. A., Lee, M. S., and D. V. Goeddel.1992. Antiviralactivity of tumor necrosis factor (TNF) is signaled through the 55-kDareceptor, type 1 TNF. J. Immunology 149:3350-3353; Rothe, J., Lesslauer,W., Lotsher, H., Land, Y., Koebel, P., Kontgen, F., Althage, A.,Zinkernagel, R., Steinmetz, M., and H. Bluethmann. 1993; Mice lackingthe tumour necrosis factor receptor 1 are resistant to TNF mediatedtoxicity but highly susceptible to infection by Listeria monocytogenes.Nature 364:798-802). All cell types specifically bound TNFα andexpressed the 55 and 75 kDa TNFα receptors. The results presented heresuggest that the anti-viral activity of TNFα was probably mediated bycritical events that occur after TNFα receptor occupancy. Furthermore,both lung epithelial and CV1 cells responded to IFN-β to restrict RSVreplication suggesting that TNFα and IFN-β can operate through separateand distinct pathways to restrict virus consistent with recent studies(Guidotti, L. G., Guilhot, S., and F. V. Chisari. 1994. Interleukin-2and alpha/beta interferon down-regulate hepatitis B virus geneexpression in vivo by tumor necrosis factor dependent and -independentpathways. J. of Virol 68:1265-1270).

TNFα can restrict virus by interfering with infection, causingcytostasis or cytolysis, or induction of genes that disrupt virusreplication (Wong, G. H. W., Kamb, A., and D. V. Goeddel.1993. Antiviralproperties of TNF. In B. Beutler, editor. Tumor Necrosis Factors: TheMolecules and Their Emerging Role in Medicine.Raven Press. Ltd, NewYork, 371-381). rTNFα mediated a minor, but significant (P<0.05),reduction in infection of lung epithelial cells, but only at low RSVdoses. At higher RSV doses, TNFα did not appear to effect infection,cell proliferation, cellular protein levels, membrane integrity, orviability in either mock or RSV infected cells but demonstrated a markeddose-dependent reduction in RSV replication.

TNFα can induce some cell types to activate TNFα/IFN-β responsive genesand can interact synergistically with IFN-β to restrict both DNA and RNAviruses (Sen, G. C., and R. M. Ransohoff. 1993. Interferon-inducedantiviral actions and their regulation. Adv. Virus Res. 42:57-101; Wong,G. H. W., Kamb, A., and D. V. Goeddel.1993. Antiviral properties of TNF.In B. Beutler, editor. Tumor Necrosis Factors: The Molecules and TheirEmerging Role in Medicine.Raven Press. Ltd, New York, 371-381). From theresults presented here, it was evident that TNFα potently restricted RSVreplication in all lung epithelial cells in a dose dependent manner and,at submaximal doses, interacted with low doses of IFN-β to essentiallyabort RSV replication in A549 cells. This interaction appeared additiverather than synergistic with RSV infected A549 cells.

TNFα and IFN-β can induce several anti-viral enzyme pathways includingthe 2', 5'A dependent RNase L to degrade viral mRNA; the double-strandedRNA-activated p68 kinase which can interfere with initiation of proteintranslation; as well as genes whose enzymatic activities have not beendefined including the IFN-78K gene which is the human equivalent of theMurine Mx gene and the ISGF2 gene (Sen, G. C., and R. M. Ransohoff.1993. Interferon-induced antiviral actions and their regulation. Adv.Virus Res. 42:57-101; Wong, G. H. W., Kamb, A., and Goeddel, D. V. 1993.Antiviral properties of TNF. In B. Beutler, editor. Tumor NecrosisFactors: The Molecules and Their Emerging Role in Medicine.Raven Press.Ltd, New York, 371-381). RNase L or activated P68 kinase should yielddecreased cellular protein levels and eventual loss of cell viability.Lung epithelial cells demonstrated no detectable change in membranepermeability or cellular viability at 24 h post-treatment with thesecytokines in both uninfected and RSV infected cells. However, recentwork by the applicants indicates the IFN and TNFα increase RNase Llevels in both human alveolar macrophages and lung epithelial cellssuggesting this pathway may be critical in restricting RSV replicationin humanlung cells. (Panuska, J. R., Rebert, N. A., Hoffmann, S. I.Anti-Respiratory Syncytial Virus Pathways in Cytokine Treated Human LungCells. Am. J. of Resp. and Crit. Care Medicine 151:A122).

Applicants further observed that TNFα, or other RSV induced products,may have a role in restricting RSV transmission between lung epithelialcells and alveolar macrophages. Lung epithelial cells were competent totransmit RSV to uninfected alveolar macrophages. In contrast, RSVinfected alveolar macrophages did not transmit RSV to uninfected lungepithelium. RSV infected alveolar macrophages neither produce defectiveinterfering virus not inactivate infectious virus (Cirino, N. M.,Panuska, J. R., Villani, A., Taraf, H., Rebert, N. A., Merolla, R.,Tsivitse, P., and I. A. Gilbert. 1993. Restricted replication ofrespiratory syncytial virus in human alveolar macrophages. J. Gen.Virol. 74:1527-1537) making it unlikely that these mechanisms couldaccount for the restricted RSV transmission.

Alveolar macrophages do release low amounts of infectious RSV whilesimultaneously producing TNF (Panuska, J. R., Midulla, F., Cirino, N.M., Villani, A., Gilbert, I. A., McFadden, E. R., and Y. T. Huang. 1990.Virus-induced alterations in macrophage production of tumor necrosisfactor and prostaglandin E₂. Am. J. Physiol (Lung Cell Mol Physiol)259:L396-L402). It is possible, that TNFα renders lung epithelial cellsresistant to the low doses of virus produced in these co-cultures.Addition of neutralizing antibodies to TNFα did not induce transmissionof RSV from alveolar macrophages to epithelial cells. However, thisresult does not preclude the possibility that TNFα was active inprotected areas, i.e. adherent sites, sequestered from the neutralizingantibody.

It is also possible that other molecules produced by alveolarmacrophages render epithelial cells resistant to RSV. Althoughapplicants do not yet understand the mechanism of uni-directionaltransmission of RSV from lung epithelial cells to alveolar macrophages,these results do suggest that virus spread through the airways does notoccur by carriage of virus by alveolar macrophages but probably resultsfrom cell-cell transmission between epithelial cells. Further studies todefine which pathways serve to restrict RSV transmission from alveolarmacrophages to lung epithelial cells are currently being investigated.

The interaction between TNFα and IFN-β to restrict RSV has a number ofphysiological and clinical implications. For example, TNFα is expressedby alveolar macrophages in vivo in children naturally infected with RSV.Although TNFα can induce lung injury in animal models, the resultspresented herein suggest that this cytokine restricts RSV replicationlocally. Indeed, murine studies have shown that local production of TNFmarkedly restricts replication of vaccinia virus (Sambhi, S. K.,Kohonen-Corish, M. R. J., and l. A. Ramshaw. 1991. Local production oftumor necrosis factor encoded by recombinant vaccinia virus is effectivein controlling viral replication in vivo. Proc Natl Acad Sci.88:40254029).

RSV is a poor inducing agent for IFN (Hall, C. B., Douglas, R. G. Jr,Simons, R. L., and J. M. Geiman.1978. Interferon production in childrenwith respiratory syncytial, influenza, and parainfluenza virusinfections. J. Pediatr. 93:28-32; Roberts, N. J. Jr, Hiscon, J., and D.J. Signs. 1992. The limited role of the interferon system response torespiratory syncytial virus challenge: analysis and comparison toinfluenza virus challenge. Microbial Pathogenesis.12:409414.) but recentstudies have shown that IFN can be delivered by aerosol to volunteers toactivate IFN responsive genes without inducing detectable changes inpulmonary functions or biopsies (Jaffe, H. A., Buhl, R., Mastrangeli,A., Holrody, K. J., Saltini, C., Czerski, D., Jaffe, H. S., Kramer, S.,Sherwin, S., and R. G. Crystal. 1991. Organ specific cytokine therapy.J. Clin. Invest 88:297-302; Martin, R. J., Boguniewicz, M., Henson, J.E., Celniker, A. C., Williams, M., Giorno, R. C., and D. Y. M. Leung.1992. The effects of inhaled interferon gamma in normal human airways.Am. Rev. Respir. Dis. 148:1677-1682; Halme, M., Maasilta, P., Mattson,K., Cantell, K. 1994. Pharmacokinetics and Toxicity of Inhaled HumanNatural Interferon-Beta in Patients With Lung Cancer. Respiration61:105-107). Furthermore, systemically administered IFN enhancesclinical improvement in RSV infected children (Sung, R. Y. T., Yin, J.,Oppenheimer, S. J., Tam, J. S., and J. Lau. 1993. Treatment ofrespiratory syncytial virus infection with recombinant interferonalfa-2a. Arch Dis. Child 69:440-442).

Consequently, by combining exogenous, nebulized IFN-β to the airwayscoupled with endogenous TNFα produced by resident alveolar macrophages,significant therapeutic benefits can be achieved in RSV infected hosts.The present invention is directed to these findings.

The invention has been described with reference to the preferredembodiment. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such modifications and alterations insofar as they come within thescope of the appended claims or the equivalents thereof.

What we claimed is:
 1. A method for treating respiratory syncytial virus(RSV) comprising administering a therapeutically effective amount ofinterferon-β (IFN-β) to lung epithelial cells harboring RSV.
 2. Themethod of claim 1, wherein said interferon-β (IFN-β) is recombinantinterferon-β.
 3. The method of claim 1, wherein said interferon-β(IFN-β) is natural interferon-β.
 4. The method of claim 3, wherein saidnatural interferon-β is human interferon-β.
 5. The method of claim 1,wherein said lung epithelial cells harboring RSV are human lungepithelial cells.
 6. A method for treating respiratory syncytial virus(RSV) comprising administering a therapeutically effective amount ofinterferon-β (IFN-β) to the lungs of a host suffering from RSV viarespiratory tract inhalation.
 7. The method of claim 6, wherein saidinterferon-β (IFN-β) is a nebulized solution of interferon-β (IFN-β). 8.The method of claim 6, wherein said interferon-β (IFN-β) is recombinantinterferon-β.
 9. The method of claim 6, wherein said interferon-β(IFN-β) is natural interferon-β.
 10. The method of claim 9, wherein saidnatural interferon-β is human interferon-β.
 11. The method of claim 6,wherein said lung epithelial cells harboring RSV are human lungepithelial cells.
 12. A method of eliciting a pharmacological effect onlung epithelial cells harboring respiratory syncytial virus (RSV)comprising the step of administering a therapeutically effective amountof interferon-β (IFN-β) to said cells.
 13. The method of claim 12,wherein said interferon-β (IFN-β) is recombinant interferon-β.
 14. Themethod of claim 12, wherein said interferon-β (IFN-β) is naturalinterferon-β.
 15. The method of claim 14, wherein said naturalinterferon-β is human interferon-β.
 16. The method of claim 12, whereinsaid lung epithelial cells harboring RSV are human lung epithelialcells.